GSM5089843: HepG2 bortezomib TRIB3 IP, 2 [HepG2-TRIB3-Flag-clone1]; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
TRIB3
Cell type
Cell type Class
Liver
Cell type
Hep G2
Primary Tissue
Liver
Tissue Diagnosis
Carcinoma Hepatocellular
Attributes by original data submitter
Sample
source_name
liver cancer cell line
cell line
HepG2-TRIB3-Flag-clone1
treatment
bortezomib 50nM 9h
chip target
TRIB3-Flag
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
Cells were dual-crosslinked with 1.5 mM ethylene glycol bis(succinimidyl succinate) (Thermo Scientific Pierce #21565) for 20 min, after which formaldehyde was added to a final concentration 1.5% and cells were incubated for another 15 min at room temperature. The crosslinking reaction was stopped by adding 0.125 M glycine. Fixed cells were treated with nuclei isolation buffer (10 mM Hepes, pH 7.9, 85 mM KCl, 0.5% NP-40, Roche Complete protease inhibitor cocktail), resuspended in ChIP lysis buffer (50 mM Tris-HCl, pH 8.0, 0.3% SDS, 10 mM EDTA, protease inhibitors) and sonicated with Bioruptor Plus (Diagenode) for 20 cycles (30 s on, 30 s off) at 4 °C with power set to high. Sonicated chromatin was diluted 3-fold in ChIP dilution buffer (5 mM Tris-HCl, pH 8.0, 250 mM NaCl, 1.1% Triton X-100, protease inhibitors) and immunoprecipitated using mouse anti-Flag M2 monoclonal antibody (Sigma #F3165) or rabbit anti-ATF4 polyclonal antibody (Santa Cruz Biotechnology sc-200) with rotation overnight at 4 °C. Immunocomplexes were isolated by Protein G sepharose beads (Amersham Biosciences) pre-blocked in 0.05% BSA. Subsequently, chromatin crosslinking was reversed and DNA was purified using QIAquick PCR Purification Kit (Qiagen). ChIP-seq libraries were prepared from 10 ng immunoprecipitated or input DNA using the NuGEN Ovation Ultralow V2 DNA-Seq library preparation kit (NuGEN Technologies). Library DNA was size selected with SPRI beads (Beckman Coulter) to retain 200-500 bp fragments. The libraries were sequenced on an Illumina NextSeq 500 using 76 bp single-end reads.